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Platelet-activating factoracetylhydrolase and other novel risk and protective factors for cardiovascular disease in systemic lupus erythematosus.

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ARTHRITIS & RHEUMATISM
Vol. 50, No. 9, September 2004, pp 2869–2876
DOI 10.1002/art.20432
© 2004, American College of Rheumatology
Platelet-Activating Factor–Acetylhydrolase and
Other Novel Risk and Protective Factors
for Cardiovascular Disease in Systemic Lupus Erythematosus
Anna Cederholm,1 Elisabet Svenungsson,2 Dominique Stengel,3 Guo-Zhong Fei,1
A. Graham Pockley,4 Ewa Ninio,3 and Johan Frostegård1
Objective. There is an important inflammatory
component to atherosclerosis and cardiovascular disease (CVD). It is therefore interesting that the risk of
CVD is high in inflammatory diseases such as systemic
lupus erythematosus (SLE). In this study, we investigated nontraditional risk factors for the development of
CVD in patients with SLE.
Methods. Twenty-six women (mean age 52 years)
with SLE and a history of CVD were compared with 26
age-matched women with SLE and no clinical manifestations of CVD (SLE controls) and 26 age-matched
healthy women (population controls). Serum levels of
several novel nontraditional risk and protective factors
were determined: heat-shock protein (HSP)–related factors (Hsp60, Hsp70, anti–human Hsp60, anti–human
Hsp70, and anti–mycobacterial Hsp65), plateletactivating factor–acetylhydrolase (PAF-AH) activity, secretory phospholipase A2 GIIA (sPLA2), and anti–
endothelial cell antibody (AECA). The intima-media
thickness and the presence of plaques in the common
carotid arteries were determined by B-mode ultrasound
as a surrogate measure of atherosclerosis.
Results. Levels of PAF-AH, but not HSP-related
factors, AECA, or sPLA2, were significantly increased in
SLE cases. Only PAF-AH discriminated between SLE
cases and SLE controls (P ⴝ 0.005). PAF-AH was
significantly associated with low-density lipoprotein
(LDL) cholesterol and total cholesterol in the SLE cases
(r ⴝ 0.50, P ⴝ 0.0093 and r ⴝ 0.54, P ⴝ 0.0045), but not
in either control group.
Conclusion. The increased levels of PAF-AH in
SLE cases and the association between PAF-AH and
LDL cholesterol adds support to the notion that
PAF-AH may promote atherothrombosis in SLE. The
role of HSPs in CVD is complex, since anti-Hsp65
appears to be associated with the presence of CVD,
whereas Hsp70 might protect against it. In this crosssectional study, levels of HSP-related factors, AECA,
and sPLA2 were not associated with CVD in SLE.
Atherosclerosis, which together with thrombosis
is a primary cause of cardiovascular disease (CVD), has
many characteristics in common with inflammatory diseases. These include an abundant production of proinflammatory cytokines and the presence of immunocompetent cells in atherosclerotic lesions (1). A growing
body of evidence indicates that patients with systemic
lupus erythematosus (SLE) are at high risk of developing CVD, and this has been interpreted as being a late
complication that has only become apparent with the
better treatments available for this patient group and the
consequential improvements in life expectancy (2,3).
We have previously reported that although traditional risk factors such as dyslipidemia were typical of
SLE-related CVD, other factors, including some
antiphospholipid-related antibodies, lipid oxidation, and
emerging inflammatory risk factors such as C-reactive
protein, were also associated with SLE-related CVD (4).
Studies of SLE-related CVD might therefore provide
Supported by the King Gustaf V 80th Birthday Fund, the
Swedish Society of Medicine, the Swedish Rheumatism Association,
the Torsten and Ragnar Söderberg Foundation, the Swedish Science
Fund, and the Swedish Heart-Lung Foundation.
1
Anna Cederholm, MD, Guo-Zhong Fei, MD, PhD, Johan
Frostegård, MD, PhD: Karolinska University Hospital, Huddinge,
Sweden; 2Elisabet Svenungsson, MD: Karolinska University Hospital, Solna, Sweden; 3Dominique Stengel, PhD, Ewa Ninio, PhD:
INSERM U525/IFR14 Coeur Muscle Vaisseaux, and Université Pierre
et Marie Curie/Faculté de Médecine Pitié-Salpêtrière, Paris, France;
4
A. Graham Pockley, PhD: University of Sheffield, Sheffield, UK.
Address correspondence and reprint requests to Johan Frostegård, MD, PhD, Department of Medicine, Karolinska University
Hospital, 141 86 Stockholm, Sweden. E-mail: johan.frostegard@ks.se.
Submitted for publication March 10, 2004; accepted April 29,
2004.
2869
2870
CEDERHOLM ET AL
important insights into the role of immunologic and
inflammatory factors in CVD, especially since laboratory animal models do not generally develop atherothrombotic disease/CVD that is typical of the condition
in humans (5).
We and other investigators have recently identified novel immunologic and inflammatory factors that
may play important roles as putative risk factors and as
causative agents in CVD and atherosclerosis. These
include Hsp60, antibodies against mycobacterial Hsp65
(6–11), antibodies against endothelial cells (AECAs)
(12), platelet-activating factor–acetylhydrolase (PAFAH) (13), and secretory phospholipase A2 (PLA2)
(14,15).
Interestingly, these novel factors are interrelated
in several ways, and their roles in atherothrombosis may
be complex and vary during different stages of disease
development. Heat-shock proteins (HSPs), or stress
proteins, are evolutionarily conserved molecules that are
expressed on activated endothelium. These proteins are
also immunogenic, and immune responses to mammalian and bacterial HSPs cross-react. In a previous study
from our laboratory, AECA was found to be associated
with anti-Hsp65 (12), and it has been reported that
anti-HSP reacts with endothelium (16). Furthermore,
oxidized LDL (ox-LDL), which the “oxidation hypothesis” suggests is a major factor in atherogenesis (17),
induces the expression of HSPs (7). PAF-AH (also
called lipoprotein-associated phospholipase A2 and
LDL-PLA2) and secretory PLA2 are also important in
the degradation of PAF and PAF-like lipids as well as in
the generation of lysophosphatidylcholine (LPC). Interestingly, PAF-like lipids and LPC are both major proinflammatory components of ox-LDL that could induce
and promote the inflammatory reaction in the vessel
wall (15,18,19). AECAs cross-react with ox-LDL and
LPC (20) and may be of major importance in vasculitis
(21).
In the present report, we provide clinical evidence that PAF-AH activity is increased in SLE patients
with CVD, whereas levels of the other nonclassic risk
factors we studied are not. The implications of these
findings are discussed.
MATERIALS AND METHODS
Study group. The SLE cases consisted of 26 women
with SLE who had 1 or more manifestations of CVD, defined
as a history of myocardial infarction (n ⫽ 7), angina (n ⫽ 9),
cerebral infarction (n ⫽ 15), or claudication (n ⫽ 4). The SLE
controls consisted of 26 age-matched women with SLE and no
clinical manifestations of CVD. The population controls con-
sisted of 26 age-matched healthy women who were recruited
randomly from the population registry. None of these subjects
had arterial disease or SLE. Details of the recruitment and
clinical characteristics of the 3 study groups have been reported previously (4).
All patients fulfilled the American College of Rheumatology (ACR) 1982 revised criteria for the classification of
SLE (22). Myocardial infarction was confirmed by electrocardiography and by an increase in the creatine kinase level.
Angina pectoris was defined as coronary insufficiency and was
confirmed by exercise stress testing. Thromboembolic, and not
hemorrhagic or vasculitic, stroke was confirmed by computed
tomography or magnetic resonance imaging. Intermittent claudication was defined as the presence of peripheral atherosclerosis and was confirmed by angiography.
The study was approved by the Ethics Committee of
Karolinska University Hospital. All subjects gave informed
consent before entering the study.
Study protocol. The investigation included a written
questionnaire, an interview, a physical examination by a rheumatologist, laboratory assessments of blood samples obtained
while the patients were fasting, and ultrasound examination of
the carotid arteries evaluated by an investigator who was
blinded to the subject’s study group. SLE disease activity was
determined using the Systemic Lupus Activity Measure
(SLAM) (23). Organ damage was determined using the Systemic Lupus International Collaborating Clinics/ACR damage
index (24). Insulin sensitivity was estimated by homeostasis
model assessment (25).
Carotid ultrasound. The right and left carotid arteries
were examined with an Acuson Sequoia duplex scanner (Siemens, Mountain View, CA), and the intima-media thickness
(IMT) was determined as described elsewhere (26). A plaque
was defined as local thickening of the intima media, with an
IMT ⬎1 mm.
Routine laboratory tests. Plasma lipoprotein concentrations, levels of homocysteine and insulin, insulin resistance,
and autoantibodies against DNA, cardiolipin, and ␤2glycoprotein I were determined by routine techniques as
described previously (4,27).
Determination of IgG and IgM AECA levels. AECAs
were detected by enzyme immunoassay as described previously, with some modifications (12). Cryopreserved pooled
human umbilical vein endothelial cells (HUVECs) at passage
2 were purchased from Cascade Biologics (Portland, OR). The
cultures were maintained at 37°C in an atmosphere of 5% CO2
in endothelial growth medium (EGM phenol red–free medium; Clonetics, San Diego, CA), containing 2% volume/
volume fetal bovine serum and supplements under humidified
conditions. HUVECs were seeded on 96-well flat-bottomed
tissue culture plates (TPP, Trasadingen, Switzerland) at a
density of 1 ⫻ 104 cells/well. After 48 hours, the cells were
washed 4 times with phosphate buffered saline (PBS), pH 7.4,
and fixed for 15 minutes at room temperature with 0.2%
glutaraldehyde in PBS. The fixed cells were washed 4 times
with washing buffer containing 0.2% bovine serum albumin
(BSA; Sigma, St. Louis, MO) in PBS. Nonspecific binding sites
were blocked for 1 hour at room temperature with 1%
weight/volume BSA–PBS and 0.1M glycine (Sigma).
Serum samples were diluted 1:50 in washing buffer,
and 100 ␮l of this dilution was added to each well. After
PAF-AH AND OTHER RISK AND PROTECTIVE FACTORS FOR CVD IN SLE
2871
Table 1. Characteristics of the study subjects at baseline*
Age, years
Disease duration, years
SLAM score
Body mass index, kg/m2
Waist-to-hip ratio
Diabetes mellitus, no. of subjects
Nephritis ever, no. of subjects
Intima-media thickness, mm
Plaque occurrence, no. of subjects
Plasma cholesterol, mmoles/liter
Plasma triglycerides, mmoles/liter
Population controls
(n ⫽ 26)
SLE cases
(n ⫽ 26)
SLE controls
(n ⫽ 26)
52.3 ⫾ 8.2
–
–
24.0 ⫾ 5.0
0.81 ⫾ 0.09
1
0
0.59 ⫾ 0.12
3
5.06 ⫾ 0.93
1.01 ⫾ 0.37
52.2 ⫾ 8.2
20.0 ⫾ 9.9
5
23.8 ⫾ 3.6
0.86 ⫾ 0.08†
3
14
0.66 ⫾ 0.15‡
17§
4.99 ⫾ 0.95
1.64 ⫾ 1.00#
52.2 ⫾ 8.2
18.5 ⫾ 9.5
6
24.0 ⫾ 3.6
0.86 ⫾ 0.09
1
9
0.60 ⫾ 0.14
10¶
5.09 ⫾ 1.14
0.96 ⫾ 0.37
* Disease activity was evaluated by the Systemic Lupus Activity Measure (SLAM). For definition of plaque occurrence,
see Patients and Methods. Values are the mean ⫾ SD, except for the SLAM scores, which are the median. Statistical
comparisons were made by analysis of variance or chi-square tests.
† P ⫽ 0.07 versus population controls.
‡ P ⫽ 0.02 versus population controls and P ⫽ 0.07 versus SLE controls.
§ P ⫽ 0.001 versus population controls and P ⫽ 0.05 versus SLE controls.
¶ P ⫽ 0.02 versus population controls.
# P ⫽ 0.0005 versus population controls and versus SLE controls.
overnight incubation at 4°C, alkaline phosphatase–conjugated
goat anti-human IgG (1:4,000 dilution) or goat anti-human
IgM (1:7,000 dilution) antibodies (both from Sigma) were
added. Absorbances at 405 nm were measured by an enzymelinked immunosorbent assay (ELISA) reader. AECA levels
(expressed as units) were calculated from a standard curve that
had been generated using a pool of positive control sera, the
concentration of which had been assigned an arbitrary value of
1,000 units.
Measurement of HSP-related factors. Serum levels of
Hsp60 and Hsp70 were determined by enzyme immunoassay
as described elsewhere (8). Briefly, 96-well microtiter plates
were coated with monoclonal antibodies to human Hsp60
(clone LK.1; Stressgen, Victoria, British Columbia, Canada) or
Hsp70 (clone C92F3A-5; Stressgen). Plates were washed and
blocked with 1% BSA. Samples were added, and bound HSP
was detected using rabbit polyclonal anti-Hsp60 or anti-Hsp70
antibody (Stressgen). Bound polyclonal antibody was detected
using alkaline phosphatase–conjugated monoclonal antibody
to rabbit immunoglobulins (Sigma), followed by p-nitrophenyl
phosphate substrate (PNPP; Sigma). The absorbance was
measured at 405 nm. Standard dose-response curves were
generated using recombinant human Hsp60 or Hsp70 (Stressgen), and the concentrations of Hsp60 and Hsp70 were
determined by reference to these standard curves using AssayZap data analysis software (Biosoft, Palo Alto, CA). The
interassay variability of the Hsp60 and Hsp70 immunoassays
was ⬍10%.
HSP antibody levels were determined as described
previously (8). Microtiter plates were coated with recombinant
human Hsp60 (Stressgen), recombinant human Hsp70 (Stressgen), or recombinant Mycobacterium bovis Hsp65 (kindly provided by Dr. M. Singh, UNDP/World Bank/World Health
Organization Special Program for Research and Training in
Tropical Diseases, Geneva, Switzerland). Plates were washed
and blocked. Samples (typical dilution 1:100) were added, and
bound antibodies were detected using alkaline phosphatase–
conjugated polyclonal goat anti-human IgA, IgG, and IgM
(Sigma) followed by PNPP substrate. Antibody concentrations
were determined by comparison with a standard curve that had
been generated using samples of predetermined high levels
that had been assigned a concentration of 1,000 arbitrary units
per milliliter.
Measurement of PLA2. Serum levels of PLA2 GIIA
were determined using a commercially available kit (human
sPLA2 ELISA, catalog no. 1666355; Boehringer, Mannheim,
Germany) according to the manufacturer’s instructions. The
sensitivity of the assay was ⬍4.4 ng/ml, with a detection range
of 1–500 ng/ml.
Measurement of PAF-AH. PAF-AH activity was measured using the trichloroacetic acid precipitation procedure, as
previously described (28). Assays were performed in 96-well
plates. Plasma that had been stored at –80°C was diluted 1:100
in 90 ␮l of PAF-AH assay buffer (pH 7.4), and 10 ␮l of 500 ␮M
3
H-labeled acetyl-PAF (mean ⫾ SD specific activity 81,000 ⫾
2,000 disintegrations per minute/nmole; DuPont NEN, Boston,
MA) was added. Samples were incubated in duplicate for 10
minutes at 37°C, and after precipitation, the radioactivity was
assessed in the supernatant. The activity of PAF-AH is expressed in nanomoles of PAF hydrolyzed/minute/ml of plasma.
A pool of control plasma (n ⫽ 10) served as an internal
standard for all measurements.
Measurement of lipids. Levels of blood lipids and
ox-LDL were measured as described previously (4); the actual
lipid and ox-LDL levels were reported in that article as well.
We used these measurements to determine their relationship
to PAF-AH.
Statistical analysis. Statistical analyses were performed using StatView software (SAS Institute, Gothenburg,
Sweden). Skewed continuous variables were logarithmically
transformed to attain a normal distribution. Study groups were
compared using analysis of variance for continuous variables
2872
CEDERHOLM ET AL
Figure 1. Distribution of platelet-activating factor–acetylhydrolase
(PAF-AH) activity in population controls (PC), systemic lupus erythematosus (SLE) cases with cardiovascular disease, and SLE controls
without cardiovascular disease. Data are shown as box plots. Each box
represents the 25th to 75th percentiles. Lines outside the boxes
represent the 10th and the 90th percentiles. Lines inside the boxes
represent the 50th percentile. Circles indicate outliers.
and the chi-square test for categorical variables. Fisher’s
protected least significant difference was used as a post hoc
test. Correlation coefficients were calculated using simple
regression or distributed variables Spearman’s rank correlation
for non-normally distributed data. The significance level was
set at P ⬍ 0.05.
RESULTS
Clinical and metabolic characteristics. Basic
clinical and metabolic characteristics of the study subjects are presented in Table 1. As previously reported
(4,27), SLE cases had elevated levels of very low-density
lipoprotein and Lp(a), decreased levels of high-density
lipoprotein (HDL) cholesterol, increased levels of acutephase reactants and erythrocyte sedimentation rates,
and elevated levels of lupus anticoagulants and homocysteine as compared with the SLE controls and the
population controls. Blood pressure, cumulative (packyears) or present numbers of cigarettes smoked, and
prevalence of diabetes mellitus did not differ significantly between the 3 groups (27).
SLE cases had taken a higher cumulative dose of
prednisolone (P ⫽ 0.05) (4,27,29); however, there was
no significant difference in the prednisolone dosage at
the time of study compared with that in the SLE
controls. Therapy with lipid-lowering agents (statins in
all cases), antihypertensive agents, low-dose aspirin,
warfarin, and azathioprine was more common among
SLE cases (4,29). There was no association between
PAF-AH activity or other nontraditional risk factors
measured in this study and medication (data not shown).
Plasma concentrations of PAF-AH, PLA2, HSPrelated factors, and AECAs. PAF-AH activity in the 3
study groups is shown in Figure 1. Levels of sPLA2,
PAF-AH, serum Hsp60 and Hsp70, and anti-Hsp60,
anti-Hsp70, anti-Hsp65, and AECAs in the 3 groups are
presented in Table 2. PAF-AH activity in SLE cases was
higher, although not statistically significantly higher, in
the SLE cases than in the SLE controls and the population controls. PAF-AH was negatively associated with
Hsp60 (r ⫽ –0.43, P ⫽ 0.037) and Hsp70 (r ⫽ –0.34, P ⫽
0.099), although the association was not significant for
Hsp70. No significant associations between PAF-AH
and the other factors we studied were noted.
Relationship between PAF-AH activity and clinical and metabolic risk factors for CVD. Correlations
between the plasma level of PAF-AH and the levels of
lipids, including lipoproteins and ox-LDL, are presented
in Table 3. A different pattern of associations was noted
between the SLE cases and the 2 control groups.
PAF-AH was strongly and positively associated with
LDL cholesterol, total cholesterol levels, and interestingly, with ox-LDL (as determined using the monoclonal
antibody EO6). However, these associations were not
present in the SLE control group; indeed, there was a
nonsignificant negative association with all 3 lipids in the
SLE control group. A significant negative association
with HDL cholesterol was also noted in the SLE control
group.
There were no associations between PAF-AH
and acute-phase reactants, anti–ox-LDL, or IMT (data
not shown) in any of the groups studied.
DISCUSSION
This study is the first to demonstrate that
PAF-AH activity in women with SLE and CVD is 30%
higher than that in age-matched women with SLE, but
without a history of CVD. There was also a trend toward
an increased level of PAF-AH in SLE cases compared
PAF-AH AND OTHER RISK AND PROTECTIVE FACTORS FOR CVD IN SLE
2873
Table 2. Novel risk and protection factors, by study group*
Hsp60, ng/ml
Mean ⫾ SD
Median
Interquartile range
Hsp70, ng/ml
Mean ⫾ SD
Median
Interquartile range
Anti-Hsp60 antibody, AU/ml
Mean ⫾ SD
Median
Interquartile range
Anti-Hsp70 antibody, AU/ml
Mean ⫾ SD
Median
Interquartile range
Anti-Hsp65 antibody, AU/ml
Mean ⫾ SD
Median
Interquartile range
sPLA2, ng/ml
Mean ⫾ SD
Median
Interquartile range
PAF-HA, nmoles/minute/ml of plasma
Mean ⫾ SD
Median
Interquartile range
IgG AECA, units
Mean ⫾ SD
Median
Interquartile range
IgM AECA, units
Mean ⫾ SD
Median
Interquartile range
SLE cases
(n ⫽ 26)
SLE controls
(n ⫽ 26)
Population controls
(n ⫽ 26)
425.9 ⫾ 311.4
348.6
333.1
500.3 ⫾ 794.7
295.6
214.8
370.03 ⫾ 255.6
330.95
350.2
239.4 ⫾ 367.9
101.9
218.8
115.4 ⫾ 120.1
76.7
84.3
176.82 ⫾ 235.5
78.85
149.2
143.1 ⫾ 146.6
94.3
182.8
92.9 ⫾ 79.4
57.13
85.2
107.07 ⫾ 139.57
60.17
99.3
6.8 ⫾ 3.8
5.4
5.1
7.1 ⫾ 3.9
6.62
3.58
7.13 ⫾ 3.58
7.13
3.47
511.8 ⫾ 255.8
496.7
354.1
494.8 ⫾ 343.5
391.6
377.4
659.51 ⫾ 395.6
530.9
539.5
16.02 ⫾ 3.08
16.6
3.2
15.98 ⫾ 2.4
16.39
2.8
14.9 ⫾ 2.63
14.3
3.75
44.0 ⫾ 16.5
40.4
18.3
34.0 ⫾ 8.8
34.4
12.4
37.6 ⫾ 10.8
36.5
11.1
702.4 ⫾ 285.5
726.0
338.0
652.6 ⫾ 179.8
643.5
221.0
614.8 ⫾ 260.7
582.0
302.2
684.6 ⫾ 314.3
740.5
505.0
635.9 ⫾ 261.7
607.5
352.0
637.9 ⫾ 259.5
609.5
304.0
* Statistical comparisons were made by analysis of variance with Fisher’s post hoc test. Only the platelet-activating
factor–acetylhydrolase (PAF-AH) level in the systemic lupus erythematosus (SLE) cases versus the SLE controls was
found to be significantly different (P ⫽ 0.005). AU ⫽ arbitrary units; sPLA2 ⫽ secretory phospholipase A2; AECA ⫽
anti–endothelial cell antibody.
with controls. Our observations could have several implications.
PAF is an important proinflammatory phospho-
lipid that is implicated in the etiology of atherosclerosis
and CVD. One reason for this is that PAF and PAF-like
lipids are involved in the proinflammatory function of
Table 3. Relationship between plasma concentrations of PAF-AH and lipids, by study group*
Population controls
Plasma triglycerides
Total cholesterol
LDL cholesterol
HDL cholesterol
Lipoprotein(a)
Oxidized LDL
SLE cases
SLE controls
r
P
r
P
r
P
0.022
0.37
0.083
0.15
0.036
⫺0.18
0.91
0.06
0.68
0.47
0.81
0.37
0.10
0.54
0.50
0.003
0.38
0.40
0.62
0.0045†
0.0093†
0.99
0.057
0.043†
0.075
⫺0.23
⫺0.18
⫺0.41
⫺0.05
⫺0.23
0.71
0.25
0.28
0.038†
0.8
0.26
* Values are Pearson’s product-moment or Spearman’s rank correlation coefficients. PAF-AH ⫽ platelet-activating factor–
acetylhydrolase; SLE ⫽ systemic lupus erythematosus; LDL ⫽ low-density lipoprotein; HDL ⫽ high-density lipoprotein.
† Statistically significant correlation.
2874
ox-LDL (18,30–32). In addition, PAF is produced by
activated platelets, monocytes, and endothelial cells and
could play an important role in endothelial activation
(30). The potential involvement of PAF in atherogenesis
has been suggested by the observation that inhibiting
PAF activity decreases the development of atherosclerosis in animal models (33).
PAF-AH is produced by cells of monocyte/
macrophage origin, as well as by T cells and mast cells,
all of which are present in atherosclerotic lesions (34).
PAF-AH degrades PAF and PAF-like lipids by hydrolyzing its acetate moiety in the sn-2 position of the
phospholipids, and its capacity to degrade PAF
prompted the proposition that PAF-AH was antiatherogenic. Indeed, animal models have shown that PAF-AH
is antiinflammatory and antiatherogenic (35). However,
PAF-AH cannot bind to LDL in mice because of
differences in amino acid composition in the 114–117
domain (36). Data from mouse models of atherosclerosis are therefore not truly reflective of the clinical
situation in this context, since in humans, 70% of
PAF-AH binds to LDL cholesterol, with the remaining
PAF-AH being transported by HDL cholesterol (37).
Instead, an increasing body of evidence indicates that
PAF-AH may be either a risk factor or a marker for
CVD, including stroke and coronary artery disease
(38–40). Our data support this proposition.
An interesting observation from the present
study is that PAF-AH activity in SLE cases was not only
associated with total cholesterol and LDL cholesterol,
but also with ox-LDL (as determined on the basis of
EO6 epitope concentration). It has been suggested that
PAF-AH is atherogenic, since it is the only lipoproteinassociated enzyme that can bind to LDL cholesterol and,
as a consequence, become trapped in the arterial wall,
within which it can induce the hydrolysis of oxidized
phospholipids. However, LPC and other oxidized phospholipid derivatives produced by PAF-AH may also be
highly atherogenic. LPC promotes not only the chemotaxis of monocytes (41), but also the activation of T cells
and their production of proinflammatory cytokines (15),
which are abundant in lesions (1). Our finding is therefore compatible with the possibility that PAF-AH promotes atherogenesis by increasing the concentration of
LPC in the arterial wall.
In sharp contrast to the situation in SLE cases,
there was no statistically significant relationship between
PAF-AH and total cholesterol, LDL cholesterol, or
ox-LDL in the SLE controls. Indeed, although not of
statistical significance, the relationship between
PAF-AH and total cholesterol, LDL cholesterol, or
CEDERHOLM ET AL
ox-LDL appeared to be a negative one. This might
indicate that PAF-AH plays a different role in the 2
groups, and it is possible that this differential role relates
to different patterns of association with lipoproteins.
However, the basis for the negative association between
PAF-AH and HDL cholesterol in SLE controls is unclear. Consistent with the findings of previous investigators (38–40), we found that PAF-AH was not associated
with common markers of inflammation in any of the
groups studied.
Circulating PLA2 enzymes, including sPLA2,
have been described in patients with atherosclerosis and
CVD. The sPLA2 enzyme promotes the modification of
phospholipids, including those present in circulating
lipoproteins. In contrast to PAF-AH, which acts only on
PAF and PAF-like lipids, sPLA2 also hydrolyzes intact
phospholipids, thereby generating lysophospholipids
and free fatty acids (42). PLA2 is present in atherosclerotic lesions (14,42,43), and may promote atherosclerosis by acting on LDL cholesterol trapped in the vascular
wall via binding to proteoglycans and inducing oxidation
of LDL (42). PLA2 may also be atherogenic and increase
the risk of CVD by inducing the formation of small,
dense LDL particles in the circulation (42). Although
PLA2 levels were not increased in the circulation of the
SLE cases in our study, this negative finding does not
necessarily rule out the possibility that sPLA2 plays a
role in artery wall damage in SLE.
The role of HSP-related factors in CVD in
general is likely to be complex. The proposition that
anti-Hsp65 is atherogenic is supported by the results of
animal experiments and clinical studies. One explanation for this finding could be that HSPs are evolutionarily conserved molecules and that anti-HSPs could
cross-react with HSPs present both on activated endothelial cells and in bacteria, including mycobacteria
(6,10,44).
Recently, HSPs, including Hsp60 and Hsp70,
have been identified in the circulation, and we have
reported that Hsp60 is significantly increased in early
CVD occurring in patients with borderline hypertension
(8). We have also demonstrated that Hsp70 appears to
protect against atherosclerosis, since high levels of
Hsp70 are associated with a decreased progression of
atherosclerosis in patients with established hypertension
(9). However, in this cross-sectional study of SLErelated CVD, HSP-related factors were not increased.
Prospective studies are necessary to elucidate the exact
role of these factors in SLE-related CVD, and it is still
possible that a defective HSP response could promote
atherothrombosis in SLE.
PAF-AH AND OTHER RISK AND PROTECTIVE FACTORS FOR CVD IN SLE
Antibodies against endothelial cells are clearly
implicated in SLE and are associated with disease
activity and vasculitis (45). Furthermore, they promote
endothelial activation by acting directly on endothelial
cells (46). AECAs have also been implicated in CVD in
the general population (12,47), and levels are increased
in SLE patients. These antibodies also cross-react with a
pivotal antigen in atherosclerosis, ox-LDL (20). However, we found that AECAs were not significantly increased in patients with SLE-related CVD in our study.
The capacity of AECAs to promote atherogenesis in
patients with more active SLE remains to be evaluated.
In conclusion, PAF-AH may play an important
role in SLE-related CVD by promoting a proinflammatory state and LDL modification of the artery wall. It
remains puzzling, however, why the generation of LPC
and oxidized short fatty acids would be more atherogenic than intact PAF and/or oxidized phospholipids. By
analogy to the increase in specific antibodies during
infection, one may also envision that the balance between these substances may be of importance (48) or
that the increase in PAF-AH is protective. Other nontraditional immune-related factors tested in this study
appear to play a lesser role in SLE-related CVD than in
CVD in the general population. Further prospective
studies are necessary to clarify whether these and other
factors are responsible for the elevated risk of CVD in
patients with SLE.
ACKNOWLEDGMENTS
We are grateful to Mikael Heimbürger for referring
patients from Huddinge University Hospital, to Jill Gustafsson
and Eva Jemseby for their help with management of the
patient cohorts and the blood sampling, to Angela Silveira and
Anders Hamsten for lipoprotein and insulin determinations, to
Joseph Witztum for help with ox-LDL determinations, and to
Kerstin Jensen-Urstad for ultrasound measurements.
5.
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15.
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18.
19.
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